When non-proliferative mammary cells of virgin mice are cultured with insulin, the cells are induced to undergo DNA synthesis. Prior to the augmentation of DNA synthesis, insulin elicits sequential stimulation of the activities of the polyamine biosynthetic enzymes, ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (S-ado-met DC) and spermidine synthase and 2-3 fold increase in the intracellular concentration of spermidine. Inhibition of spermidine formation by methyl glyoxal bis(guanylhydrazone), MCBG, an inhibitor of S-ado-met DC prevents an increase in DNA synthesis without affecting RNA and protein synthesis. Exogenous spermidine at physiological concentrations, however, reverses the MGBG inhibition of DNA synthesis to a normal level. Spermidine in the absence of insulin does not stimulate DNA synthesis. These results, together with previous findings indicate that spermidine plyas an essential role for the regulation of both cell proliferation and milk protein synthesis during the hormone dependent development of mammary epithelium.